Hypomethylation of CTCFL promoters as a noninvasive biomarker in plasma from patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third deadliest cancer in the world with high morbidity and poor prognosis. CTCFL (CCCTC-binding factor like) is a member of the cancer testis antigen (CTA) family with oncogenic properties. To demonstrate whether the hypomethylation of CTCFL promoters in plasma could be used as a noninvasive biomarker to predict poor prognosis of HCC, we extracted cell-free DNA from the plasma and detected the methylation status of CTCFL in 43 HCC, 5 liver cirrhosis and 6 benign lesion samples using methylation specific PCR (MSP). Our study indicated that the hypomethylation of CTCFL promoters in HCC plasma samples (60.4%) was significantly different from that in benign lesion plasma samples (16.7%) with a p-value of 0.043. Analysis of clinicopathological data showed that the methylation status of CTCFL promoters was significantly correlated with microvascular involvement (MVI) (p=0.001) and postoperative recurrence (p=0.031). Furthermore, clinical prognosis data of 347 HCC patients from The Cancer Genome Atlas (TCGA) database displayed that the hypomethylated group had worse overall survival than the hypermethylated group (p=0.0056). In conclusion, we provide evidence that the hypomethylation of CTCFL promoters in cell-free DNA is a biomarker for monitoring HCC patients, which can be used as a noninvasive prediction index for tumor recurrence and provide the individualized decision-making for clinicians.

The prognostic value of combination of CD90 and OCT4 for hepatocellular carcinoma after curative resection.

CD90 has been identified as a candidate marker of cancer stem cells (CSCs) for HCC, whereas it also has been considered as a marker for tumor-associated fibroblasts (CAFs). OCT4, as a key transcription factor required to maintain pluripotency of human embryonic stem cell and cancer cells, has been characterized to be involved in malignant transformation and tumorigenesis of various cancers. This study aimed to examine expression patterns of CD90 in HCC and investigate whether combination of both CD90 and OCT4 could provide a more powerful predictor for prognosis of HCC than either one alone.CD90 and OCT4 were examined by immunohistochemistry. The relationship between CD90/OCT4 expression and clinicopathologic characteristics was analyzed. The correlation between CD90/OCT4 expression and overall survival and disease-free survival was determined with Kaplan-Meier analysis.CD90 was found mainly expressed in tumor-associated CAFs and OCT4 was mainly expressed in tumor cells. The expression of CD90 and OCT4 in HCC was significantly higher than in adjacent non-tumor and normal liver tissues. CD90 expression was correlated with pathological grade, satellite lesion, PVTT and recurrence. OCT4 expression was correlated with pathological grade, tumor size and recurrence. Data demonstrated no correlation between CD90 and OCT4. High expression of CD90 or OCT4 predicts a poor prognosis. Furthermore, combination of both CD90 and OCT4 provides a more sensitive predictor for prognosis of HCC than either marker alone.CD90 and OCT4 are both independent and reliable biomarker for predicting prognosis of HCC patients after hepatic resection. Our results indicated the accuracy of prediction can be enhanced by their combination.

Flk-1+ mesenchymal stem cells aggravate collagen-induced arthritis by up-regulating interleukin-6.

The immunomodulatory ability of mesenchymal stem cells (MSCs) may be used to develop therapies for autoimmune diseases. Flk-1(+) MSCs are a population of MSCs with defined phenotype and their safety has been evaluated in Phase 1 clinical trials. We designed this study to evaluate whether Flk-1(+) MSCs conferred a therapeutic effect on collagen-induced arthritis (CIA), an animal model of rheumatic arthritis, and to explore the underlying mechanisms. Flk-1(+) MSCs, 1-2 x 10(6), were injected into CIA mice on either day 0 or day 21. The clinical course of arthritis was monitored. Serum cytokine profile was determined by cytometric bead array kit or enzyme-linked immunosorbent assay. Flk-1(+) MSCs and splenocytes co-culture was conducted to explore the underlying mechanisms. Flk-1(+) MSCs did not confer therapeutic benefits. Clinical symptom scores and histological evaluation suggested aggravation of arthritis in mice treated with MSCs at day 21. Serum cytokine profile analysis showed marked interleukin (IL)-6 secretion immediately after MSC administration. Results of in vitro culture of splenocytes confirmed that the addition of Flk-1(+) MSCs promoted splenocyte proliferation and increased IL-6 and IL-17 secretion. Moreover, splenocyte proliferation was also enhanced in mice treated with MSCs at day 21. Accordingly, MSCs at low concentrations were found to promote lipopolysaccharide-primed splenocytes proliferation in an in vitro co-culture system. We propose that Flk-1(+) MSCs aggravate arthritis in CIA model by at least up-regulating secretion of IL-6, which favours Th17 differentiation. When Flk-1(+) MSCs are used for patients, we should be cautious about subjects with rheumatoid arthritis.

Using human adipose tissue-derived mesenchymal stem cells as salvage therapy for hepatic graft-versus-host disease resembling acute hepatitis.

A 43-year-old woman with chronic hepatic graft-versus-host disease (GVHD) who failed previous immunosuppressive therapy with cyclosporine and prednisone was treated with tacrolimus starting on day 165 after allogeneic hematopoietic stem cell transplantation. Fifteen days later, tacrolimus was discontinued because of progressive deterioration of renal function. However, after changing treatment to human adipose tissue-derived mesenchymal stem cells (AMSC), we observed rapid and complete resolution of hepatic GVHD and renal toxicity. We concluded that it is worthwhile to administer AMSC as a treatment for common hepatic GVHD, particularly for atypical cases presenting as acute hepatitis.

Favorable response to human adipose tissue-derived mesenchymal stem cells in steroid-refractory acute graft-versus-host disease.

There is no consistently effective therapy for patients with steroid-refractory acute graft-versus-host disease (GVHD). Various alternative approaches have been tested, including antithymocyte globulin, mycophenolate mofetil (MMF), pentostatin, and monoclonal antibodies; however, they have only been modestly successful. The purpose of our study was to evaluate the efficacy of human adipose-tissue-derived mesenchymal stem cells (AMSC) as salvage therapy for steroid-refractory acute GVHD. Six patients with steroid-refractory grades III-IV acute GVHD received IV infusions of AMSC. The AMSC dose was 1.0x10(6)/kg. No side effects were noted after the AMSC infusions. Five patients were treated once and one patient twice. Two patients received AMSC from haplo-identical family donors and four from unrelated mismatched donors. Acute GVHD disappeared completely in five of six patients, four of whom are alive after a median follow-up of 40 months (range, 18-90 months) after the initiation of AMSC therapy. All four surviving patients are in good clinical condition and in remission of their hematological malignancy. Two patients died-one with no obvious response to AMSC died of multiorgan failure and one a relapse of leukemia. These results suggested that AMSC is a promising treatment for severe steroid-resistant acute GVHD.

Research progress about effects of myocardial enzyme and troponin on uremia with acute left ventricular failure.

OBJECTIVE: We studied the diagnostic value of CK-MB and troponin (cTnI) in uremia with acute left ventricular failure patients. PATIENTS AND METHODS: We enrolled 130 uremia patients with maintenance hemodialysis (MHD) and divided them into two groups: (i) the observation group with patients suffering from acute left ventricular failure (n=30) and (ii) the control group which contained cases without acute left ventricular failure (n=100). We verified CK-MB, cTnI, serum creatinine, blood urea nitrogen, pro-BNP and LVEF levels at 6 h, 12 h, 24 h, 48 h, 72 h, 7 d and 14 d after the attack and carried out 1-year follow-up to compare total mortality and cardiogenic mortality. RESULTS: Our results showed that CK-MB and cTnI levels in the observation group were significantly higher than those in the control group (p<0.05). CK-MB and cTnI in the observation group increased into platform stage slowly with no peak or downtrend. They were in a linear pattern in the control group. Comparison of SCr and BUN in two groups at different time points produced no statistically significant differences (p>0.05). Pro-BNP levels in the hospital as well as 1 month, 6 months and 12 months follow-ups were higher than those in the control group, and differences were of statistical significant (p<0.05). While in hospital LVEF level in the observation group was higher than that in the other group, differences regarding 1 month, 6 months and 12 months follow-up between two groups had no statistical significance (p>0.05). Total mortality and cardiogenic mortality in the observation group were higher than those in the control group, and differences were statistically significant (p<0.05). CONCLUSIONS: CK-MB, cTnI, SCr, BUN, pro-BNP and LVEF were independent risk factors for total mortality while CK-MB, cTnI and pro-BNP were independent risk factors for cardiogenic mortality.

Observation of the efficacy of radiofrequency catheter ablation on patients with different forms of atrial fibrillation.

OBJECTIVE: To study the efficacy and safety of radiofrequency catheter ablation (RFCA) in patients with different forms of atrial fibrillation. PATIENTS AND METHODS: By retrospective analysis, we summarize 720 cases, where patients diagnosed with atrial fibrillation in our hospital were treated with RFCA from February 2010 to October 2014. Among the cases, 425 were diagnosed with paroxysmal atrial fibrillation and 295 with non-paroxysmal atrial fibrillation (including persistent atrial fibrillation and permanent atrial fibrillation). All patients were followed up until June 2015 to compare and analyze the differences in operation success rates, complications and recurrence rates. RESULTS: 395 cases (92.9%) of paroxysmal atrial fibrillation and 253 cases (85.8%) with non-paroxysmal atrial fibrillation were subject to surgery and followed up. The age of onset, disease course, underlying diseases, left atrial diameter and combined anti-arrhythmics of patients with paroxysmal atrial fibrillation were lower than those of patients with non-paroxysmal atrial fibrillation, and the differences were statistically significant (p < 0.05). The success rate of the first ablation was higher than that of non-paroxysmal atrial fibrillation. Procedure time, procedure method, complications and recurrence rate of patients with paroxysmal atrial fibrillation were lower than those of non-paroxysmal atrial fibrillation group, and the differences were statistically significant (p < 0.05). When we compared apoplexy and heart failure caused by atrial fibrillation in the two groups, the difference was not statistically significant (Apoplexy: p = 0.186; Heart failure: p = 0.170). CONCLUSIONS: The individual ablation success rate was higher for paroxysmal atrial fibrillation, and long-term follow-up showed that the occurrence of apoplexy and heart failure was not different from the non-paroxysmal atrial fibrillation group.

miR-302 regulates pluripotency, teratoma formation and differentiation in stem cells via an AKT1/OCT4-dependent manner.

Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine, but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here, we determined that the downregulation of miR-302 suppresses the teratoma formation, hampers the self-renewal and pluripotency, and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1, which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly, we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation.

Establishment and properties of fetal dermis-derived mesenchymal stem cell lines: plasticity in vitro and hematopoietic protection in vivo.

Human mesenchymal stem cells (hMSCs) are excellent candidates for ex vivo gene transfer and cell therapy in various systems. However, hMSCs are mortal somatic cells, and thus invariably enter an irreversible growth arrest after a finite number of cell divisions in culture. It has been proposed that this is due to telomere shortening. In this study, pGRN145 plasmid containing human telomerase reverse transcriptase (hTRT) was introduced into fetal dermis-derived hMSCs. Single-cell clones positive for telomerase activity and hTRT mRNA were selected and expanded. Single-cell-derived hTRT(+) cells could be expanded rapidly in vitro and passaged up to 70 doublings without showing senescence. FACScan flow cytometer showed that hTRT(+) cells were positive for CD29, CD44, CD105, and CD166, while CD31, CD45, CD34, vWF, and HLA-DR were negative. Under suitable conditions, hTRT(+) cells have the ability of multiple lineage differentiation, including bone, fat, and nerve. Furthermore, transplantation of hTRT(+) cells could protect NOD/SCID mice from lethal irradiation. Thus, these cells may be an ideal cell source for promoting hematopoietic recovery after radiotherapy.

miR-216a rescues dexamethasone suppression of osteogenesis, promotes osteoblast differentiation and enhances bone formation, by regulating c-Cbl-mediated PI3K/AKT pathway.

Osteoporosis is a disease marked by reduced bone mass, leading to an increased risk of fractures or broken bones. Bone formation is mediated by recruiting mesenchymal stem cells (MSCs). Elucidation of the molecular mechanisms that regulate MSC differentiation into osteoblasts is of great importance for the development of anabolic therapies for osteoporosis and other bone metabolism-related diseases. microRNAs (miRNAs) have been reported to have crucial roles in bone development, osteogenic differentiation and osteoporosis pathophysiology. However, to date, only a few miRNAs have been reported to enhance osteogenesis and regulate the suppressive effect of glucocorticoids on osteogenic differentiation. In this study, we discovered that miR-216a, a pancreatic-specific miRNA, was significantly upregulated during osteogenic differentiation in human adipose-derived MSCs (hAMSCs). The expression of miR-216a was positively correlated with the expression of bone formation marker genes in clinical osteoporosis samples. Functional analysis demonstrated that miR-216a can markedly promote osteogenic differentiation of hAMSCs, rescue the suppressive effect of dexamethasone (DEX) on osteogenic differentiation in vitro and enhance bone formation in vivo. c-Cbl, a gene that encodes a RING finger E3 ubiquitin ligase, was identified as a direct target of miR-216a. Downregulation of c-Cbl by short hairpin RNAs can mimic the promotion effects of miR-216a and significantly rescue the suppressive effects of DEX on osteogenesis. Pathway analysis indicated that miR-216a regulation of osteogenic differentiation occurs via the c-Cbl-mediated phosphatidylinositol 3 kinase (PI3K)/AKT pathway. The recovery effects of miR-216a on the inhibition of osteogenesis by DEX were attenuated after blocking the PI3K pathway. Thus, our findings suggest that miR-216a may serve as a novel therapeutic agent for the prevention and treatment of osteoporosis and other bone metabolism-related diseases.

MicroRNA-197 influences 5-fluorouracil resistance via thymidylate synthase in colorectal cancer.

PURPOSE: The response rate of first-line fluoropyrimidine-based regimens for metastatic colorectal cancer (mCRC) is generally less than 50 %. The down-regulation of miR-197 in colorectal cancer cells after exposure to 5-fluorouracil might be related to the mechanism of resistance to fluoropyrimidine-based chemotherapy. So we investigated the regulatory mechanism of miR-197 on 5-FU sensitivity. METHODS: Dual luciferase reporter gene construct and dual luciferase reporter assay were used to identify the target of miR-197. TYMS expression was evaluated by immunohistochemistry staining. 5-Fu resistance of colorectal cancer cell lines was detected by MTS assay. The expression of miR-197 was detected by real time PCR. RESULTS: A luciferase assay and western blot analysis confirmed that miR-197 directly binds to and negatively regulates TYMS expression. Overexpressing miR-197 could increase the sensitivity of colorectal cancer cells to 5-fluorouracil (5-FU). The expression of miR-197 negatively correlated with TYMS expression in cancerous tissues from patients with stage IV colorectal cancer. CONCLUSION: miR-197 mediates the response of colorectal cancer cells to 5-FU by regulating TYMS expression.

Changes in tissue metals after cadmium intoxication and intervention with chlorpromazine in male rats.

Cadmium (Cd), one of the most dangerous heavy metals, has a very similar ionic radius to calcium (Ca). The interference of cadmium in calcium homeostasis may play an important role in cadmium toxicity. Recent reports indicate that calmodulin (CaM) inhibitors such as trifluoperazine and chlorpromazine (CPZ) could protect rodents against cadmium toxicity. It was also reported that pretreatment of mice with zinc (Zn) could reduce the adverse effects induced by cadmium. The aim of this study is to determine whether Cd changes the balance of other essential metals such as Zn and copper (Cu) in rat tissues, and whether CPZ can reverse these changes which are induced by cadmium intoxication. Adult male Sprague-Dawley (SD) rats were injected intraperitoneally (i.p.) with cadmium chloride (CdCl2) (0.2, 0.4, 0.8 mg Cd/kg body weight) alone and 0.4 mg Cd/kg in association with CPZ (5 mg/kg) daily for a week. The control animals were injected with normal saline only. The results showed that the cadmium content in the liver, kidney and testis increased significantly with a dose-response relationship. Cadmium treatment markedly increased the Zn and Ca content in some of the tissues. Hepatic and renal metallothionein (MT) increased significantly after cadmium intoxication. CPZ treatment, however, reduced cadmium content in liver, but not blood and kidney. CPZ seemed to decrease the content of MT in liver and significantly increase the amounts of MT in kidney. These data suggest that the intervention of cadmium with tissue essential metals may play a role in cadmium toxicity in rats, and calmodulin inhibitors to some extent can reduce the adverse effect of cadmium by decreasing the cadmium load in tissues and reversing the unbalance of essential metals.

Association of cystatin C-based glomerular filtration rate with SYNTAX score in patients with diabetes.

AIM: Serum cystatin C has been proposed as a better marker of glomerular filtration rate than serum creatinine. SYNTAX score (SXscore) can accurately reflect the severity of coronary artery disease (CAD). However, the association between Cystatin C-based glomerular filtration rate (eGFRcys) and SXscore in patients with diabetes has never been reported. METHODS: We prospectively included 656 consecutive patients with diabetes who were angiographically diagnosed with CAD from January 2010 to December 2011. Renal function was assessed by eGFRcys. SXscore was calculated using SXscore algorithm. Ordinal logistic regression and Pearson correlation were used to analyze the association between eGFRcys and SXscore. RESULTS: Patients with renal dysfunction were older, more often female, more likely to have a history of hypertension and less tobacco use when compared with those patients with normal renal function. Age, sex, SBP, DBP, fasting glucose, HbA1c, TC, LDL, HDL, TG, BMI and CRP were not different among SXscore tertile groups. Incidence of hypertension, hyperlipidemia, family history and tobacco use were similar among these groups. Correlation analysis suggested that eGFRcys was negatively correlated with SXscore (R=-0.255, P<0.001). Ordinal logistic regression showed that eGFRcys was an independent predictor of SXscore (ß=-0.027, P<0.001). CONCLUSIONS: eGFRcys was an independent predictor of SXscore in patients with diabetes. This might help explain the increased risk of CVD events and mortality in patients with renal dysfunction. Further prospectively multiple centre studies are required to better quantify this finding.

Human adipose tissue-derived adult stem cells can lead to multiorgan engraftment.

Recent studies have demonstrated the existence of a population of adipose tissue-derived adult stem cells that can undergo multilineage differentiation in vitro; however, it is unclear whether these cells maintain their multilineage differentiation in vivo. The objective of the present study was to examine the in vivo characteristics and behavior of a potential population of human adipose tissue-derived adult stem cells. Herein, we demonstrate that human adipose tissue-derived adult stem cells differentiate into the epithelium of the gastrointestinal tract, liver, and bronchi, and an endothelial lineage after transplantation into irradiated nonobese mice with diabetes or severe combined immunodeficiency. These findings may contribute to clinical tissue repair after injury.

Resolution of refractory chronic autoimmune thrombocytopenic purpura following mesenchymal stem cell transplantation: a case report.

Herein we have reported the case of a man with chronic autoimmune thrombocytopenia (AITP) refractory to conventional therapy who underwent T-cell-depleted autologous peripheral blood stem cell (PBSC) transplantation. A complete, steroid-independent, platelet remission was obtained, but unfortunately he subsequently relapsed after 3 months. Finally, we administered human adipose tissue-derived mesenchymal stem cells (AMSC) with resolution to the AITP.

Human mesenchymal stem cells inhibit cancer cell proliferation by secreting DKK-1.

Mesenchymal stem cells (MSCs) have an inhibitory effect on tumor proliferation, but the precise mechanisms are not fully understood. Here, we identified DKK-1 (dickkopf-1), secreted by MSCs and acting as a negative regulator of WNT signaling pathway, to be one of the molecules responsible for the inhibitory effect. When DKK-1 was neutralized by anti-DKK-1 antibodies, or when the expression of DKK-1 was downregulated by RNA interference (RNAi), the inhibitory effects of MSCs on K562 cell proliferation were attenuated. We also provide evidence that the expression of DKK-1 by MSCs is regulated by NANOG, a transcriptional factor ubiquitously expressed in some stem cells. Using the Cellmax artificial capillary modules that eliminate the immunosuppressive properties of MSCs, we further showed that MSCs were able to inhibit proliferation of K562 cells in a humoral microenvironment. Meanwhile, we recapture this effect of MSCs on primary leukemic hematopoietic progenitors from patients. MSCs probably have a general inhibitory effect on their neighboring cells, including malignant cells, en route to achieving tissue homeostasis.

Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity.

beta(1)-integrin engagement on normal (NL) CD34(+) cells increases levels of the cyclin-dependent kinase inhibitor (cdki), p27(Kip), decreases cdk2 activity, and inhibits G(1)/S-phase progression. In contrast, beta(1)-integrin engagement on chronic myelogenous leukemia (CML) CD34(+) cells does not inhibit G(1)/S progression. We now show that, in CML, baseline p27(Kip) levels are significantly higher than in NL CD34(+) cells, but adhesion to fibronectin (FN) does not increase p27(Kip) levels. p27(Kip) mRNA levels are similar in CML and NL CD34(+) cells and remain unchanged after adhesion, suggesting posttranscriptional regulation. Despite the elevated p27(Kip) levels, cdk2 kinase activity is similar in CML and NL CD34(+) cells. In NL CD34(+) cells, >90% of p27(Kip) is located in the nucleus, where it binds to cdk2 after integrin engagement. In CML CD34(+) cells, however, >80% of p27(Kip) is located in the cytoplasm even in FN-adherent cells, and significantly less p27(Kip) is bound to cdk2. Thus, presence of BCR/ABL induces elevated levels of p27(Kip) and relocation of p27(Kip) to the cytoplasm, which contributes to the loss of integrin-mediated proliferation inhibition, characteristic of CML.

Gene therapy for chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is characterized by a balanced translocation that leads to the formation of the the BCR-ABL fusion gene. Although autografts can prolong the life of CML patients, patients relapse owing to malignant cells that persist in the graft and the host. This review discusses various experimental strategies that target the BCR-ABL gene or gene products that are downstream of it. Various strategies have been adopted to block BCR-ABL at the gene, mRNA and protein level. One promising strategy involves the cotransduction of a patient's hematopoietic stem cells (HSCs) with anti-BCR-ABL antisense sequences and a drug resistance gene. This might allow for the elimination of any residual disease in the graft or host by chemotherapy while rendering any drug-resistant, malignant CML HSCs functionally normal.